Absorptive enterocytes in the small intestine contain four homologous lipid-binding proteins which represent over 5% of the total soluble cellular protein. These proteins are termed ileal lipid-binding protein, intestinal fatty acid-binding protein, "liver" fatty acid-binding protein, and cellular retinol-binding protein II. They are proposed to serve distinct functional roles in absorption, transport and metabolism of different classes of dietary and biliary lipids. Because of their different specificities, these proteins may ultimately represent excellent targets for the rational design of agents to selectively reduce the intestinal absorption of saturated and unsaturated fatty acids, bile salts, and cholesterol. However, the factors that control molecular recognition in this family of proteins has not yet been established. The main objective of this research is to identify the structural, dynamic and energetic determinants of ligand-binding affinity and specificity for the four distinct lipid-binding proteins from the small intestine. The specific aims of the proposed research are: (i) to characterize the binding pro mutant and chimeric lipid- binding proteins, (ii) to establish sequence-specific 1H, 13C, and 15N NMR assignments and determine the solution structures for several mutant proteins with altered ligand specificities, and (iii) to design and synthesize novel ligands that bind with high affinity to act as inhibitors. The feasibility of the proposed experiments is firmly established by preliminary results, including the high-yield expression and purification of natural abundance and isotope-enriched proteins, the development of biophysical assays for probing lipid-protein interactions, the generation of mutants with altered specificity, and the collection and assignment of triple-resonance 3-D NMR data with excellent sensitivity and resolution. A high intake of dietary fat has been associated with an increased risk of atherosclerosis and colon cancer. A detailed knowledge of the molecular factors governing intestinal fat absorption and homeostasis will ultimately lead to more precise dietary recommendations and novel pharmacologic strategies to prevent these prevalent and life-threatening diseases. In particular, a detailed study of the molecular determinants of lipid-binding specificity, stoichiometry and affinity in the lipid-binding proteins from intestine will contribute fundamentally important information for this purpose.